C e n t r e d e R e c e r c a e n To x i c o l o g i a

PRODUCED NON-INTENTIONALLY MANUFACTURED

extrapolation

antropogenic (diesel)

natural (fire, nucleation)

existing (CB, developed ad TiO2) hoc, modifyed

• Biomedical uses• Biomedical uses:

– drug delivery

– sensors or diagnostic systems working from inside the cell

– structures for implants (scaffolds), may afford the possibility of imitating “porous” organs, or p y g p g ,presenting capillary structures, as the liver

– etcetc.

• In the scientific language:g g

– Statistic concept (probability)

Exposure

Real harmHazard

– Assessment of exposure and effects: incertainity

Modulating factors

Assessment of exposure and effects: incertainity

• Risk zero does not exist• Risk zero does not exist

• … and perhaps is even not desirable

• Risk / benefit balance• Risk / benefit  balance

• Exposure– Non‐ intentional– Non‐ intentional

• Atmospheric (primary or secondary), traffic (60%), combustion processes, volcanoes, erosion, marine spray, etc..

Manufactured particles– Manufactured particles• Respiratory

• Dermal

• Oral

• 1. How does exposure occur?

• 2. What or who is exposed?

• 3. How much exposure occurs? When and where does it

• occur?

• 4 How does exposure vary?• 4. How does exposure vary?

• 5. How uncertain are exposure estimates?

• 6. What is the likelihood that exposure will occur?

• Present needsU f l ti l t t h i– Use of novel particle measurement techniques 

– Development of personal samplers to determine personal exposure to NP and their agglomerates 

– Development of a model describing the dispersion and transformation of NP and their agglomerates

• Non‐soluble Nanoparticles can stay for years in the lungs, GI‐tract or brain; they are less well taken up by “professional”tract or brain; they are less well taken up by  professional  macrophages of the defense system but interact with cells of the epithelium, the interstitial tissue and vascular cells the epithelium, the interstitial tissue and vascular cellsallowing pro‐inflammatory reactions of these cells which usually do not see any particles. 

• In addition, Nanoparticles can bind to proteins or translocateinto the circulation and reach secondary target organs like liver, spleen, kidneys, heart and brain

• Pulmonary EffectsI d i fl t ff t (f di l )– Increased inflammatory effects (free radicals)

– Very high load tumors  (rat)– Effects on reticulum–endothelial system

• Cardiovascular effects and hemocompatibility– Hemolysis, coagulation– CV effects known but poorly understood

• Central Nervous System– Hypertension– Allergic Encephalomyelitis– Harm to BBB– Oxidative stress

• Nanoparticles show special characteristics that may modulate its toxic potential:

• increased absorption / through unusual waysp g y• cross barriers• penetrate into otherwise scarcely accessible organs• interact with subcellular targets in special and poorly knowninteract with subcellular targets in special and poorly known conditions

• Drugs delivered at nano scale:• Drugs delivered at nano‐scale:• reach new tissue, cell or molecular targets• and they do it in scarcely predictable conditions

• However, lower equipotent doses and a more accurate vectorizationshould contribute to a decrease of toxicity

• its assessment present special challenges. – Its behaviour toward biological systems is influenced by many factors including 

• shape, • Coatingsi h t h t i di t ib ti• inherent heterogeneous size distribution. 

– In NPs the internalization process is a crucial event and should be characterizedand should be characterized.

• In that complex context toxicological methods adapted to these new materials should be developedthese new materials should be developed.

• Within the frame of NANOSOST, we have tested the toxicity of 4 kinds of nanoparticles (NPs): 

– gold (AuNP) diameter: 10,45nm

– gold coated by hyaluronic acid (HA‐AuNP) diameter: 30 g y y ( )nm

– cobalt ferrite coated by silane (Si‐CoFe2O4 NP) diameter:  17 nm

– cobalt ferrite coated by silane and gold  (Au‐Si‐CoFe2O4

NP) diameter: 45 nm. 

• Acute toxicity in rats by intraperitoneal (IP) administration. Thespecific aim of the study was to determine a DL by the Up&Downspecific aim of the study was to determine a DL50 by the Up&Downprotocol (OECD 425) and compare these results with the valuesobtained in parallel for gold (III) chloride (HAuCl4) and CoFe2O4 insolutionsolution.• Clinical signs,• necropsy,• hematological parameters and• histopathological exam (kidney, liver, spleen and lung)

• Biodistribution by TEM (liver, spleen, kidney and lung)

• ICP‐MS of different tissues (liver, spleen, pancreas, lymph node,kidney, lung and brain) at 14 days from the administration wasassessed.assessed

LD 50 Low Lim LD 50 Up Lim LD 50

Au salts 106,56 42,63 266,41

Cobalt ferrite salts 121,9 48,76 304,74

Not differences in hematologicalNot differences in hematologicalvaluesNot differences in biochemicalparametersparametersSome differences in histophatologicalevaluation but it’s because pH

0,2

AuNP

0,05

0,1

0,15

ppm

0

0 15'  30'  1h  3h  6h  12h 24h 48h  72h

2

AuNP con HA

1

1,5

ppm

0,006

0,007

NP ferrita cobalt0

0,5

0 15'  30'  1h  3h  6h  12h 24h 48h  72h

0 001

0,002

0,003

0,004

0,005

ppm

0

0,001

0 15'  30'  1h  3h  6h  12h 24h 48h  72h

• Cytotoxicity : LLC‐PK1 and Hep2 (representing potential target organs following systemic administration) by means of WST‐1 and LDH methods.

• Mutagenicity: Bacterial mutagenicity was assessed in 4 S l ll th hi i d 1 E h i hi li t i ithSalmonella thyphimurium and 1 Escherichia coli strain with and without metabolic activation according to Ames protocol. 

• Uptake of the NPs by cells was studied by fluorescence microscopy and ICP‐MSmicroscopy and ICP MS.

• The samples were not mutagenic in the assayed conditions

1100

1200S9 - *

400

450

500

S9 -S9

*

S. typhimurium TA100 ( 881)

400

500

600

700

800

900

1000

1100

Rev

ertie

ntes

/pla

ca

S9 +

*

E. coli WP2 uvrA

200

250

300

350

400

ertie

ntes

/pla

ca

S9 +

0

100

200

300

Blanco E D C B A Control +

Concentración µg/placa)

R

nsnsns nsns

ns ns nsns ns

0

50

100

150

200

Rev

e

nsnsns ns ns ns ns nsns ns

*

Blanco E D C B A Control +

Concentración (µg/placa)

• No conclusive results could be obtained nor with LDH nor with WST‐1

Hep G2. WST1 tiempo 48hLLC-PK1. LDH48horas

120

60,000

80,000

100,000

120,000

bilid

ad (%

)

40

60

80

100

iber

ació

n LD

H (%

)

0,000

20,000

40,000

ultiv

o ce

l

[NP]

1

[NP]

2

[NP]

3

[NP]

4

[NP]

5

[NP]

6

[NP]

7

[NP]

8

[NP]

9

AP 5

0mM

X100

1%

Via b

0

20

40

tónX

100 1

%APAP 50

mM

[NP]1

[NP]2

[NP]3

[NP]4

[NP]5

[NP]6

[NP]7

[NP]8

[NP]9

L

• Cytotoxicity will be studied using a cellular testing method which does not employ absorbance measurements (i e: Colony Forming Efficiency

C(-

)C

C(+

)APA

Trito

n X

Tritó

C+AP

not employ absorbance measurements (i.e: Colony Forming EfficiencyTest” (CFE))

NPAu-HA/cél.

200000

120000

140000

160000

180000

200000

-HA

20000

40000

60000

80000

100000

nº N

PAu

0

20000

Media NPAu/cel 65816 163556

6h 24h

Quantification by ICP AES

Internalization by Confocal microscopy of AuNP coated with HA

Quantification by ICP-AES

Thanks for your attention

Joaquín de LapuenteJoaquín de Lapuentejlapuente@pcb.ub.cat

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